Luminal Breast Cancer Cell Lines Research Articles

Abstract PO2-24-09: Prolonged Effects of Hypoxia in Luminal Breast Cancer Cells Promotes an Aggressive Phenotype

Abstract Aidan Moriarty Mentor: Dr. Min Yu Hypoxic Memory in Breast Cancer Cells Metastasis is the main cause of tumor-related mortality in breast cancer patients. Improving our understanding of the metastatic cascade is imperative to improve patient outcomes. Prior research indicates cancer cells with increased metastatic potential have acquired adaptive properties induced by factors in the tumor microenvironment (TME), including hypoxia. However, previous hypoxia research has been primarily focused on signaling changes that occur when cancer cells are in the hypoxic TME and does not account for long-term changes that may occur once cancer cells re-enter normoxia, such as when they metastasize. Our research in patient-derived circulating tumor cell (CTCs) lines and breast cancer cell lines has indicated that certain transcriptional changes are maintained after cells leave hypoxia, and that these post-hypoxic CTCs maintain a higher metastatic potential compared to cells not exposed to hypoxia. Taking an un-biased approach to define changes in hypoxia-exposed breast cancer cells upon reoxygenation, we performed bulk RNA-Seq on luminal breast cancer cell lines in various oxygen conditions over time. We found that a subset of genes that were differentially expressed in hypoxic conditions were maintained as up- or down-regulated after cells were reoxygenated, indicating a “hypoxic memory”. Additionally, a subset of genes were differentially expressed in the post-hypoxic cells compared to normoxia controls, demonstrating novel and prolonged gene expression changes induced by hypoxia exposure. We performed functional assays and observed that this post-hypoxic state is associated with a more aggressive phenotype in vitro. Current research in our lab is on-going to understand the epigenetic changes that may be responsible for this hypoxic memory to further understand the consequences of hypoxic memory in CTC biology and metastasis. Finally, we have designed a dual-reporter hypoxia-tracing system to validate our findings in vivo. This research will improve our understanding of the prolonged influence of hypoxia on metastatic potential in breast cancer cells and has the potential to inform future therapeutic strategies. Citation Format: Aidan Moriarty, Oihana Iriondo, Desirae Mecenas, Yilin Li, Yonatan Amzaleg, Remi Klotz, Min Yu. Prolonged Effects of Hypoxia in Luminal Breast Cancer Cells Promotes an Aggressive Phenotype [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-09.

Abstract 6837: Prolonged effects of hypoxia in luminal breast cancer cells

Abstract Metastasis is the main cause of tumor-related mortality in breast cancer patients. Improving our understanding of the metastatic cascade is imperative to improve patient outcomes. Prior research indicates cancer cells with increased metastatic potential have acquired adaptive properties induced by factors in the tumor microenvironment (TME), including hypoxia. However, previous hypoxia research has primarily focused on signaling changes that occur when cancer cells are in the hypoxic TME and does not account for long-term changes that may occur once cancer cells re-enter normoxia, such as when they metastasize. Our research in patient-derived circulating tumor cell (CTCs) lines and breast cancer cell lines indicates that certain transcriptional changes are maintained after cells leave hypoxia, and that these post-hypoxic cells maintain a higher metastatic potential compared to cells not exposed to hypoxia. Taking an un-biased approach to define changes in hypoxia-exposed breast cancer cells upon reoxygenation, we performed bulk RNA-Seq on post-hypoxic luminal breast cancer cell lines compared to normoxic controls cultured either in adherent or suspension conditions, the latter to mimic the physiological context of CTCs. We found that a subset of genes that were differentially expressed in hypoxic conditions were maintained as up- or down-regulated after cells were reoxygenated, indicating a “hypoxic memory”. Additionally, a subset of differentially expressed genes were unique to the post-hypoxic cells, demonstrating both prolonged and novel gene expression changes induced by hypoxia exposure. We also performed functional assays and observed that this post-hypoxic state is associated with a more aggressive phenotype. Current research in our lab is on-going to understand the epigenetic changes that may be responsible for this hypoxic memory. Overall, this research will improve our understanding of the prolonged consequences of hypoxia on CTC biology and metastasis. Citation Format: Aidan Moriarty, Oihana Iriondo, Yonatan Amzaleg, Remi Klotz, Min Yu. Prolonged effects of hypoxia in luminal breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6837.

Role of hydroxymethylglutharyl-coenzyme A reductase in the induction of stem-like states in breast cancer

PurposeDe novo synthesis of cholesterol and its rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stem-like states in tumors remains unclear.MethodsA compiled public database from breast cancer (BC) patients was analyzed with the web application SurvExpress. Cell Miner was used for the analysis of HMGCR expression and statin sensitivity of the NCI-60 cell lines panel. A CRISPRon system was used to induce HMGCR overexpression in the luminal BC cell line MCF-7 and a lentiviral pLM-OSKM system for the reprogramming of MCF-7 cells. Comparisons were performed by two-tailed unpaired t-test for two groups and one- or two-way ANOVA.ResultsData from BC patients showed that high expression of several members of the cholesterol synthesis pathway were associated with lower recurrence-free survival, particularly in hormone-receptor-positive BC. In silico and in vitro analysis showed that HMGCR is expressed in several BC cancer cell lines, which exhibit a subtype-dependent response to statins in silico and in vitro. A stem-like phenotype was demonstrated upon HMGCR expression in MCF-7 cells, characterized by expression of the pluripotency markers NANOG, SOX2, increased CD44 +/CD24low/ −, CD133 + populations, and increased mammosphere formation ability. Pluripotent and cancer stem cell lines showed high expression of HMGCR, whereas cell reprogramming of MCF-7 cells did not increase HMGCR expression.ConclusionHMGCR induces a stem-like phenotype in BC cells of epithelial nature, thus affecting tumor initiation, progression and statin sensitivity.

TRPS1 modulates chromatin accessibility to regulate estrogen receptor alpha (ER) binding and ER target gene expression in luminal breast cancer cells.

Common genetic variants in the repressive GATA-family transcription factor (TF) TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over 80 percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes estrogen receptor alpha (ER) to redistribute in the genome. ER redistribution leads to both repression and activation of dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the genomic distribution of ER, both activating and repressing transcription of genes related to cancer cell fitness.

Characterizing the Inflammatory Profile of Neutrophil-Rich Triple-Negative Breast Cancer.

Breast cancer (BC) is one of the most common types of cancer in women in the United Arab Emirates. Immunogenic tumours, such as triple-negative breast cancer (TNBC), show increased neutrophil infiltration, which is associated with poor prognosis and limited efficacy of immunotherapy. This study aims to investigate in vitro the bidirectional effect of neutrophils on metastatic TNBC (MDA-MB-231) compared to less-metastatic luminal breast cancer (MCF-7) cell lines. We found that BC cells or their conditioned medium (CM) reduced the viability of neutrophil-like cells (HL60). This was supported by increased cellular stress and NETosis in differentiated HL60 cells (dHL60) upon exposure to MDA-MB-231 compared to MCF-7-CM using nucleic acid staining essays. Flow cytometry showed comparable expression of inflammatory markers by polymorphonuclear cells (PMN) when treated with MDA-MB-231-CM and standard polarizing cocktails. Furthermore, MDA-MB-231-CM triggered an inflammatory pattern with evidence of stronger adhesion (CD62L) and degranulation (CD11b and CD66b) phenotypes. The proinflammatory polarization of dHL60 by MDA-MB-231-CM was additionally confirmed by the elevated CD54 expression, myeloperoxidase, and CD11b protein levels, which matched an increased transwell migratory capacity. In conclusion, BC might use neutrophils to their benefit through NETosis and complement system activation, which makes this crosstalk a potential mechanism for understanding tumour progression.

The Expression of Hsa-Mir-1225-5p Limits the Aggressive Biological Be-haviour of Luminal Breast Cancer Cell Lines.

Numerous genetic and biological processes have been linked to the func-tion of microRNAs (miRNAs), which regulate gene expression by targeting messenger RNA (mRNA). It is commonly acknowledged that miRNAs play a role in the development of disease and the embryology of mammals. To further understand its function in the oncogenic process, the expression of the miRNA profile in cancer has been investigated. Despite being referred to as a noteworthy miRNA in cancer, it is unknown whether hsa-miR-1225-5p plays a part in the in vitro progression of the luminal A and luminal B subtypes of breast cancer. We proposed that a synthetic hsa-miR-1225-5p molecule be expressed in breast cancer cell lines and its activity be evaluated with the aim of studying its function in the development of luminal breast cancer. In terms of the typical cancer progression stages, such as proliferation, survival, migration, and invasion, we investigated the role of hsa-miR-1225-5p in luminal A and B breast cancer cell lines. Additionally, using bioinformatics databases, we thoroughly explored the target score-based prediction of miRNA-mRNA interaction. Our study showed that the expression of miR-1225-5p significantly inhibited the in vitro growth of luminal A and B breast cancer cell lines. The results were supported by a bioinformatic analysis and a detailed gene network that boosts the activation of signaling pathways required for cancer progression.

Progestins and breast cancer hallmarks: The role of the ERK1/2 and JNK pathways in estrogen receptor positive breast cancer cells

Progestins used in hormonal contraceptives and menopausal hormone therapy (MHT) have been linked to increased breast cancer risk. Whether the association holds for all progestins is unclear and the underlying mechanisms remain poorly understood. We directly compared the effects of four progestins (medroxyprogesterone acetate (MPA), norethisterone acetate (NET-A), levonorgestrel (LNG) and drospirenone (DRSP)) to each other and the natural progestogen progesterone (P4) on selected cancer hallmarks. To provide mechanistic insight into these effects, we assessed the role of the progesterone receptor (PR), and the extracellular signal-related kinase (ERK1/2) and c-Jun N terminal (JNK) signaling pathways. We showed that the increased proliferation of the luminal T47D breast cancer cell line by P4 and all progestins, albeit to different extents, was inhibited by PR knockdown and inhibition of both the ERK1/2 and JNK pathways. While knockdown of the PR also blocked the upregulation of MKI67 and CCND1 mRNA expression by selected progestogens, only a role for the ERK1/2 pathway could be established in these effects. Similarly, only a role for the ERK1/2 pathway could be confirmed for progestogen-induced colony formation, whereas both the ERK1/2 and JNK pathways were required for cell migration in response to the three older progestins implicated in the etiology of breast cancer, MPA, NET-A and LNG. Together our results show that all the progestins elicit their effects on cell proliferation via a mechanism requiring the PR, ERK1/2 and JNK pathways. While the ERK1/2 and JNK pathways are also required for increased cell migration by the older progestins, only a role for the ERK1/2 pathway could be established in their effects on colony formation. Notably, the cytoplasmic PR was not needed for activation of the ERK1/2 pathway by the progestogens. Given that DRSP showed significantly lower proliferation than MPA and NET-A, and that it had no effect on breast cancer cell migration and colony formation, hormonal formulations containing the newer generation progestin DRSP may provide a better benefit/risk profile towards breast cancer than those containing the older generation progestins.

A molecular toolbox to study progesterone receptor signaling

Progesterone receptor (PR) signaling is required for mammary gland development and homeostasis. A major bottleneck in studying PR signaling is the lack of sensitive assays to measure and visualize PR pathway activity both quantitatively and spatially. Here, we develop new tools to study PR signaling in human breast epithelial cells. First, we generate optimized Progesterone Responsive Element (PRE)-luciferase constructs and demonstrate that these new reporters are a powerful tool to quantify PR signaling activity across a wide range of progesterone concentrations in two luminal breast cancer cell lines, MCF7 and T47D. We also describe a fluorescent lentiviral PRE-GFP reporter as a novel tool to visualize PR signaling at the single-cell level. Our reporter constructs are sensitive to physiological levels of progesterone. Second, we show that low background signaling, and high levels of PR expression are a prerequisite for robustly measuring PR signaling. Increasing PR expression by transient transfection, stable overexpression in MCF7 or clonal selection in T47D, drastically improves both the dynamic range of luciferase reporter assays, and the induction of endogenous PR target genes as measured by qRT-PCR. We find that the PR signaling response differs per cell line, target gene and hormone concentration used. Taken together, our tools allow a more rationally designed approach for measuring PR signaling in breast epithelial cells.

Downregulation of MALAT1 in triple-negative breast cancer cells

BackgroundMALAT1 is one of the most abundant nuclear long non-coding RNAs, which has been found to be elevated in various types of cancers. However, conflicting reports on MALAT1 in breast cancer cell lines challenge understanding of MALAT1's involvement in breast cancer progression. AimMeasurement of normalized relative quantity (NRQ) of MALAT1 transcripts in cell lines representing triple-negative breast cancer (TNBC) and luminal breast cancer. Materials and methodsThe studies were performed using cell lines representing luminal breast cancer (T47D, MCF-7), TNBC (MDA-MB-468, CAL-51, MDA-MB-231), and MCF-10A cell line of normal breast epithelial cells. Total RNA was isolated from six independent cell cultures of each line, treated with DNase I, and used to synthesize complementary DNA, which was used in quantitative real-time PCR (qPCR) assays. Four MALAT1 fragments and reference genes CCSER2, ANKRD17, PUM1, GAPDH were amplified. ResultsGeometric means of the NRQ of MALAT1 in breast cancer cell lines had the shortest 95% confidence intervals when CCSER2 was used for normalization. MALAT1 major transcript levels thus estimated in TNBC cell lines were found to be statistically significantly reduced compared to levels in both MCF-10A cells and luminal breast cancer cell lines, while MALAT1 minority splice variants were found to be increased in almost all breast cancer cell lines. ConclusionCCSER2-normalized qPCR results indicate MALAT1 downregulation in cell lines representing the more aggressive breast cancer subtype compared to both the normal breast epithelial cell line and the estrogen receptor-positive breast cancer cell lines.

TWIST1 Upregulation Is a Potential Target for Reversing Resistance to the CDK4/6 Inhibitor in Metastatic Luminal Breast Cancer Cells.

Cyclin-dependent kinase (CDK) 4/6 inhibitors have significantly improved progression-free survival in hormone-receptor-positive (HR+), human-epidermal-growth-factor-receptor-type-2-negative (HER2-) metastatic luminal breast cancer (mLBC). Several studies have shown that in patients with endocrine-sensitive or endocrine-resistant LBC, the addition of CDK4/6 inhibitors to endocrine therapy significantly prolongs progression-free survival. However, the percentage of patients who are unresponsive or refractory to these therapies is as high as 40%, and no reliable and reproducible biomarkers have been validated to select a priori responders or refractory patients. The selection of mutant clones in the target oncoprotein is the main cause of resistance. Other mechanisms such as oncogene amplification/overexpression or mutations in other pathways have been described in several models. In this study, we focused on palbociclib, a selective CDK4/6 inhibitor. We generated a human MCF-7 luminal breast cancer cell line that was able to survive and proliferate at different concentrations of palbociclib and also showed cross-resistance to abemaciclib. The resistant cell line was characterized via RNA sequencing and was found to strongly activate the epithelial-to-mesenchymal transition. Among the top deregulated genes, we found a dramatic downregulation of the CDK4 inhibitor CDKN2B and an upregulation of the TWIST1 transcription factor. TWIST1 was further validated as a target for the reversal of palbociclib resistance. This study provides new relevant information about the mechanisms of resistance to CDK4/6 inhibitors and suggests potential new markers for patients' follow-up care during treatment.

FAM84B promotes breast cancer tumorigenesis through activation of the NF-κB and death receptor signaling pathways

Breast cancer (BC) occurs predominantly in women and leads to numerous deaths every year. The identification of effective therapeutic targets will benefit BC patients and increase the likelihood of finding a cure. Family with similar sequence 84, member B (FAM84B) has been implicated in the progression of many kinds of cancers, but its function in BC remains to be explored. In this study, online database analysis revealed that FAM84B expression was higher in BC patient tissues, especially in luminal BC tissues, than in the corresponding normal tissues; furthermore, increased FAM84B expression was related to poor prognosis. Additionally, western blot (WB) analysis revealed that the FAM84B protein was highly expressed in luminal BC cell lines compared to normal and basal-like BC cell lines. Moreover, clinical BC patient tissues were collected and subjected to WB and immunohistochemical (IHC) analyses, and the results showed that FAM84B was expressed mainly in luminal BC samples. Therefore, to determine the function of FAM84B in luminal BC cells, luminal BC cell lines with FAM84B knockout and overexpression were generated. In addition, the functions of FAM84B were evaluated in vitro (via cell proliferation, wound healing, colony formation and invasion assays) and in vivo (via a subcutaneous xenograft experiment), and the results showed that FAM84B regulated cell proliferation but not cell invasion. Furthermore, the results of RNA sequencing analysis in ZR-75–1 FAM84B knockout and FAM84B-overexpressing cells showed that FAM84B could affect the TNF signaling pathway. Subsequently, WB analysis of death receptor signaling and immunofluorescence (IF) analysis of NF-κB p65 localization revealed that FAM84B affected death receptor signaling and promoted NF-κB p65 nuclear entry. In conclusion, we found that FAM84B promotes luminal BC tumorigenesis through the activation of the NF-κB and death receptor signaling pathways.

Irreversible HER2 inhibitors overcome resistance to the RSL3 ferroptosis inducer in non-HER2 amplified luminal breast cancer.

Ferroptosis, a form of programed cell death, can be promoted by inhibitors of the xCT transporter (erastin) or GPX4 (RSL3). We found that GPX4, but not the xCT transporter, is selectively elevated in luminal breast cancer. Consistent with this observation, the majority of luminal breast cancer cell lines are exquisitely sensitive to RSL3 with limited sensitivity to erastin. In RSL3-resistant, but not sensitive, luminal breast cancer cell lines, RSL3 induces HER2 pathway activation. Irreversible HER2 inhibitors including neratinib reversed RSL3 resistance in constitutively RSL3-resistant cell lines. Combination treatment with RSL3 and neratinib increases ferroptosis through mitochondrial iron-dependent reactive oxygen species production and lipid peroxidation. RSL3 also activated replication stress and concomitant S phase and G2/M blockade leading to sensitivity to targeting the DNA damage checkpoint. Together, our data support the exploration of RSL3 combined with irreversible HER2 inhibitors in clinical trials.

Parabens Promote Protumorigenic Effects in Luminal Breast Cancer Cell Lines With Diverse Genetic Ancestry.

One in 8 women will develop breast cancer in their lifetime. Yet, the burden of disease is greater in Black women. Black women have a 40% higher mortality rate than White women, and a higher incidence of breast cancer at age 40 and younger. While the underlying cause of this disparity is multifactorial, exposure to endocrine disrupting chemicals (EDCs) in hair and other personal care products has been associated with an increased risk of breast cancer. Parabens are known EDCs that are commonly used as preservatives in hair and other personal care products, and Black women are disproportionately exposed to products containing parabens. Studies have shown that parabens impact breast cancer cell proliferation, death, migration/invasion, and metabolism, as well as gene expression in vitro. However, these studies were conducted using cell lines of European ancestry; to date, no studies have utilized breast cancer cell lines of West African ancestry to examine the effects of parabens on breast cancer progression. Like breast cancer cell lines with European ancestry, we hypothesize that parabens promote protumorigenic effects in breast cancer cell lines of West African ancestry. Luminal breast cancer cell lines with West African ancestry (HCC1500) and European ancestry (MCF-7) were treated with biologically relevant doses of methylparaben, propylparaben, and butylparaben. Following treatment, estrogen receptor target gene expression and cell viability were examined. We observed altered estrogen receptor target gene expression and cell viability that was paraben and cell line specific. This study provides greater insight into the tumorigenic role of parabens in the progression of breast cancer in Black women.

Abstract 5994: INX-315, a potent and selective CDK2 inhibitor, demonstrates robust antitumor activity in CCNE1-amplified cancers

Abstract Cyclin-dependent kinases (CDK) are a family of serine/threonine kinases that heterodimerize with regulatory subunits called cyclins to drive cell cycle progression. Uncontrolled cellular proliferation is a hallmark of cancer commonly driven by dysregulated kinase activity of specific CDK family members, including cyclin-dependent kinase 2 (CDK2). Aberrant CDK2 activity most frequently occurs through amplification of CCNE1 and/or overexpression of its protein product cyclin E1, which is a canonical binding partner of CDK2. Overexpression of cyclin E1 is observed in many solid tumors including in patients with high grade serous ovarian cancer (HGSOC), gastric cancer, and ER-positive breast cancer patients whose tumors have progressed on a prior CDK4/6 inhibitor regimen. Selective inhibition of CDK2 is thus a compelling therapeutic approach to regain cell cycle control. Here, we report preclinical data supporting the development of INX-315 for patients with cancers in which proliferation is CDK2-dependent. INX-315 is a potent inhibitor of CDK2/cyclin E (0.6 nM biochemical IC50) with high selectivity over other CDK family members in both biochemical and intracellular NanoBRET assays. In CCNE1-amplified human ovarian and gastric cancer cell lines, INX-315 potently inhibited Rb phosphorylation, induced a G1 cell cycle arrest, and inhibited proliferation. INX-315 also showed potent anti-proliferative activity in luminal breast cancer cell lines that had been cultured in CDK4/6 inhibitor (+/- anti-estrogen therapy) for prolonged periods to the point of developing drug resistance. While these cell lines did not readily respond to either CDK4/6 or CDK2 inhibition alone, combination treatment again suppressed Rb phosphorylation, accompanied by G1 arrest and a senescent-like phenotype. Lastly, in CCNE1-amplified xenograft models of ovarian and gastric carcinomas, INX-315 inhibited Rb-phosphorylation and induced tumor regression. These data demonstrate INX-315 to be a potent and selective CDK2 inhibitor that may benefit patients with CDK2/cyclin E driven cancers. Citation Format: Alec G. Trub, John E. Bisi, Catherine Dietrich, Michael Taylor, Jay C. Strum, Shom Goel, Patrick J. Roberts. INX-315, a potent and selective CDK2 inhibitor, demonstrates robust antitumor activity in CCNE1-amplified cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5994.

Chalcone-3 Inhibits the Proliferation of Human Breast Cancer MDA-MB-231 Cell Line.

Chalcone-3 has been shown to be cytotoxic and selective against luminal subtype breast cancer cell lines, which are suspected to occur through the mechanism of epidermal growth factor receptors (EGFR) inhibition. However, the cytotoxic effect has never been tested on cell strains from patients with triple negative breast cancer (TNBC), where EGFR expression is known to increase. This study aimed to identify the role of chalcone-3 in one of the downstream targets of EGFR as an antiproliferative agent. Chalcone-3 was examined for its effect on proliferation in human breast cancer MDA-MB-231 cell lines. The percentage of proliferation inhibition was analyzed using methyl-thiazol tetrazolium assay. Flow cytometry was used to analyze the population of cell cycle distribution and the expression of cyclin-D1 and pEGFR. Chalcone-3 inhibited the proliferation of MDA-MB-231 cells in a dose and time-dependent manner with an IC50 value of 17.98±6.36 µg/mL by inducing cell cycle arrest at the G2/M phase. Flow cytometry assays showed that chalcone-3 significantly reduced the expression of pEGFR and cyclin-D1, contributing to cell cycle arrest. Chalcone-3 might have potential as an anti-proliferative drug to treat TNBC.

GRHL2-controlled gene expression networks in luminal breast cancer

Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells.6_BaCm7NaLGTK4K5qg2XWLVideo

RF22 | PMON05 Parabens Promote Pro-Tumorigenic Effects in Luminal Breast Cancer Cell Lines With Diverse Genetic Ancestry

Abstract In the United States, Black women are 39% more likely to die from breast cancer compared to White women (AACR Cancer Disparities Progress Report 2020). Furthermore, Black women are at a higher risk of developing breast cancer under the age of 40 than any other racial or ethnic group. While the underlying cause of these disparities is multifactorial, exposure to endocrine disrupting chemicals (EDCs) in hair and personal care products has been associated with increased risk of breast cancer. Parabens are known EDCs that are commonly used as preservatives in hair and other personal care products. The Environmental Working Group scores methylparaben (MP), propylparaben (PP), and butylparaben (BP) moderate (MP) to high (BP and PP) on a hazardous scale of chemicals found in personal care products. Furthermore, these parabens are restricted in personal care products with set maximum concentration standards in Japan, the European Union, and Southeast Asia. Studies have shown that parabens impact breast cancer cell proliferation, death, migration/invasion, and metabolism, as well as gene expression in vitro. However, these studies were conducted using cell lines of European ancestry; to date, no studies have utilized breast cancer cell lines of West African ancestry to examine effects of parabens. We hypothesize that, like what has been shown for breast cancer cell lines with European ancestry, parabens will promote pro-tumorigenic effects in breast cancer cell lines with West African ancestry. To test this hypothesis, we treated MCF-7 (European ancestry) and HCC1500 (West African ancestry) luminal breast cancer cell lines with biologically relevant doses of either MP, PP, or BP. We also co-treated with the estrogen receptor (ER) antagonist ICI 182,780 to determine whether observed effects are mediated by ER. We found BP increases HCC1500 cell viability, but not MCF-7 cell viability. This effect was not blocked by co-treatment with ICI 182,780. We also observed that BP and PP, but not MP, increased expression of estrogen-regulated genes in both MCF-7 and HCC1500. The increase in gene expression was reduced with co-treatment of ICI 182,780 in both cell lines, suggesting that these effects are ER-mediated. Preliminary studies suggest that parabens promote migration of both cell lines, with a potentially greater effect on HCC1500 cells. Taken together, these data demonstrate that parabens, particularly BP and PP, promote pro-tumorigenic effects in diverse luminal breast cancer cell lines. These findings have future translational relevance as we are part of a community-led initiative called Bench to Community. Bench to Community brings together basic researchers in endocrinology and social-behavioral scientists, community stakeholders including breast cancer survivors to develop interventions to reduce adverse exposures to EDCs in hair and other personal care products in Black women. Presentation: Sunday, June 12, 2022 12:42 p.m. - 12:47 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Functional Characterization of lncRNA152 as an Angiogenesis-Inhibiting Tumor Suppressor in Triple-Negative Breast Cancers.

This study identifies lncRNA152 as an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC by upregulating the expression of the tumor suppressor RBM47. As such, lncRNA152 may serve as a biomarker to track aggressiveness of breast cancer, as well as therapeutic target for treating TNBC.

In vitro radiosensitization of breast cancer with hypoxia-activated prodrugs.

KP167 is a novel hypoxia‐activated prodrug (HAP), targeting cancer cells via DNA intercalating and alkylating properties. The single agent and radiosensitizing efficacy of KP167 and its parental comparator, AQ4N, were evaluated in 2D and 3D cultures of luminal and triple negative breast cancer (TNBC) cell lines and compared against DNA damage repair inhibitors. 2D normoxic treatment with the DNA repair inhibitors, Olaparib or KU‐55933 caused, as expected, substantial radiosensitization (sensitiser enhancement ratio, SER0.01 of 1.60–3.42). KP167 induced greater radiosensitization in TNBC (SER0.01 2.53 in MDAMB‐231, 2.28 in MDAMB‐468, 4.55 in MDAMB‐436) and luminal spheroids (SER0.01 1.46 in MCF‐7 and 1.76 in T47D cells) compared with AQ4N. Significant radiosensitization was also obtained using KP167 and AQ4N in 2D normoxia. Although hypoxia induced radioresistance, radiosensitization by KP167 was still greater under 2D hypoxia, yielding SER0.01 of 1.56–2.37 compared with AQ4N SER0.01 of 1.13–1.94. Such data show KP167 as a promising single agent and potent radiosensitiser of both normoxic and hypoxic breast cancer cells, with greater efficacy in TNBCs.

Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers.

Breast cancer is the most frequent malignancy in women worldwide. Basal-like breast cancer (BLBC) is the most aggressive form of this disease, and patients have a poor prognosis. Here, we present data suggesting that the Hippo-transcriptional coactivator with PDZ-binding motif (TAZ) pathway is a key driver of BLBC onset and progression. Deletion of Mob1a/b in mouse mammary luminal epithelium induced rapid and highly reproducible mammary tumorigenesis that was dependent on TAZ but not yes-associated protein 1 (YAP1). In situ early-stage BLBC-like malignancies developed in mutant animals by 2 wk of age, and invasive BLBC appeared by 4 wk. In a human estrogen receptor+ luminal breast cancer cell line, TAZ hyperactivation skewed the features of these luminal cells to the basal phenotype, consistent with the aberrant TAZ activation frequently observed in human precancerous BLBC lesions. TP53 mutation is rare in human precancerous BLBC but frequent in invasive BLBC. Addition of Trp53 deficiency to our Mob1a/b-deficient mouse model enhanced tumor grade and accelerated cancer progression. Our work justifies targeting the Hippo-TAZ pathway as a therapy for human BLBC, and our mouse model represents a powerful tool for evaluating candidate agents.